The main goal of this proposal is to generate extravillous trophoblast (EVT) from human embryonic stem cells (hESC) after they have been treated with BMP4 (BMP-hESC) and use them as a model to study EVT emergence, migration, and invasiveness, as well as intrinsic and extrinsic factors that influence these properties. This project has high significance because EVT ultimately become the invasive trophoblast (TR) cells of human placenta that penetrate maternal blood vessels and enhance blood flow. While invasion of the uterine decidua and the proximate inner third of the myometrium by EVT is a characteristic feature of a healthy pregnancy, shallow invasion is associated with placental pathologies, among them pre-eclampsia (PE) and intra-uterine growth retardation (IUGR). PE is a pregnancy-specific syndrome that affects ~ 3 % of pregnancies and is responsible for 15 % of pre-term births and an estimated 50,000 deaths per year worldwide. It is characterized by maternal hypertension, edema, and proteinuria, conditions that in the more serious, early onset disease can manifest as soon as 20 weeks of pregnancy. Aim 1 will establish and characterize a hESC-derived cell culture model for the study of EVT. This aim is based on the hypothesis that the sub-population of HLA-G positive BMP-hESC that migrates through a Matrigel-coated matrix under high oxygen (O2) conditions are EVT homologs. HLA-G positive cells that remain associated with the BMP-hESC colonies under low O2 conditions represent a precursor population of non- invasive EVT whose properties will be distinct from the invasive population captured under high O2 conditions. Aim 2 will examine the ability of the EVT derived from BMP-hESC to invade through cross-linked, relatively stiff matrices, such as Type I collagen, and to interact with microcapillaries cultured within such matrices. Aim 3 will examine a possible model for PE in which up-regulation of stable HIF1 alpha (HIF1A) protein is hypothesized to counteract the effects of high O2 during in vitro development of BMP-hESC and retard development into invasive EVT. If time permits or the HIF1A hypothesis proves incorrect, we would plan to study the effects of knockdown of at least one transcription factor that has been implicated in EVT emergence and differentiation, namely ASCL2. Aim 4 will attempt to re-create EVT from infants born to mothers with PE by generating induced pluripotent stem cells (iPSC) from discarded umbilical cord and converting these pluripotent cells to CT, EVT and other lineages by the BMP4 approach. The properties of these cells can then be compared with EVT generated from cord cells from pregnancies not complicated by PE. The hypothesis underpinning this aim is that some forms of PE are initiated by genetically-based placental pathology, and these abnormalities will be manifested in the phenotype of EVT generated from umbilical cord-derived iPSC. Together, these aims will bring innovative and new approaches to the study of human TR cells and to examining the basis of placental pathologies, especially PE. They will create the first in vitro model that makes possible the study of both the emergence and invasiveness of EVT cells, and the first model to study these early events directly in TR from pregnancies complicated by PE.